Solid-Phase Organic Syntheses
.pdf90 N-Fmoc-aminooxy-2-chlorotrityl Polystyrene Resin
min 1); collected in a Buchner funnel; and successively washed with DMF (10 mL), DCM (20 mL), and hexane (5 mL), and dried in vacuo overnight.
Amount of resin product recovered 232 mg.
Fmoc-substitution (note 5) 0.81 mmol g 1, 97% acylation efficiency.
The derivatized resin product (100 mg, 0.08 mmol) was suspended in DCM (6 mL) for 30 min, after which 0.06 mL TFA was added and the resultant suspension was gently stirred for 15 min at ambient temperature. The suspension was filtered, the spent resin was washed with DCM (5 mL) and DCM:MeOH (1:1, 5 mL), and the filtrate was evaporated to dryness in vacuo to give the title compound (29 mg, 90%) as white crystalline solid. RPHPLC analysis (G1) showed the exclusive presence (> 98%) of Fmoc-Phe-NHOH (Rt ¼ 6.8 min).
m / z (ES(þ)) calculated, 403.17 (MHþ ), observed, 403.4 (MHþ ).
H (250 MHz, [2H]6-DMSO) 2.88 (1H, m, Phe C H), 3.98– 4.21 (4H, m, Phe C H & Fmoc CHCH2), 7.13–7.43, 7.66, 7.87 (13H, m, Phe Ar. CHs & Fmoc Ar. CHs), 7.76 (1H, d, J 8.7 Hz, Phe N H), 8.92 (1H, br s, NH), 10.75 (1H, br s, OH).
N-(9-Fluorenylmethoxycarbonyl)valinyl Hydroxamic Acid
N-Fmoc-aminooxy-2-chlorotrityl polystyrene (115 mg, 1.00 mmol g 1, 0.115 mmol) was treated as outlined above and Fmocdeprotected using 20% v/v piperidine in DMF (10 min, 2.5 mL min 1). The resin was then washed with DMF (10 min, 2.5 mL min 1) after which excess DMF was removed. Fmoc-Val-OH (204 mg, 0.6 mmol), HOAt (81 mg, 0.6 mmol) and HATU3 (232 mg, 0.6 mmol) were dissolved in DMF (1.2 mL) and DIEA
Procedures 91
(209 ml, 1.2 mmol) then added. After ca. 1 min, the mixture was added to the resin and the reaction suspension gently agitated at room temperature for 24 h (note 7). The resin was then washed with DMF (10 min, 2.5 mL min 1), collected in a Buchner funnel, and successively washed with DMF (10 mL), DCM (20 mL) and hexane (5 mL), and dried in vacuo overnight.
Amount of resin product recovered was 124 mg.
Fmoc-substitution (note 5) 0.78 mmol g 1, 87% acylation efficiency.
The derivatised resin product was suspended in DCM (6 mL) for 30 min, after which TFA (0.06 mL) was added and the resultant suspension was gently stirred for 15 min at ambient temperature. The suspension was filtered, the spent resin washed with DCM (5 mL) and DCM:MeOH (1:1, 5 mL), and the combined filtrate was evaporated to dryness in vacuo to afford the title compound (25 mg, 73%) as a white crystalline solid. RPHPLC analysis (G1) showed the exclusive presence (>98%) of Fmoc-Val-NHOH (Rt ¼ 5.1 min).
m / z (ES(þ)) calculated 355.17 (MHþ ), observed, 355.0 (MHþ ), 377.2 (MþNaþ ).
H (250 MHz, [2H]6-DMSO) 0.87 (3H, d, J 6.97 Hz, Val C H3), 0.91 (3H, d, J 6.87 Hz, Val C H3), 1.94 (1H, m, Val C H), 3.66 (1H, t, J 8.78 Hz, Fmoc CH), 4.17– 4.33 (3H, m, Val C H & Fmoc CH2), 7.29–7.45, 7.76, 7.87 (9H, m, NH & Fmoc Ar. CHs), 7.53 (1H, d, J 9.0 Hz, Val N H), 10.68 (1H, br s, OH).
N-(4-Methoxybenzenesulphonyl)leucyl Hydroxamic Acid
N-Fmoc-aminooxy-2-chlorotrityl polystyrene (100 mg, 1.00 mmol g 1, 0.1 mmol) was treated as outlined above and Fmoc-depro-
92 N-Fmoc-aminooxy-2-chlorotrityl Polystyrene Resin
tected using 20% v/v piperidine in DMF (10 min, 2.5 mL min 1). The resin was then washed with DMF (10 min, 2.5 mL min 1) after which excess DMF was removed. Fmoc-Leu-OH (212 mg,
0.6 mmol), |
HOAt |
(81 mg, |
0.6 mmol), |
and |
HATU3 |
(232 mg, |
|
0.6 mmol) |
were dissolved |
in DMF (1.2 mL); and |
DIEA was |
||||
(209 mL, 1.2 mmol) |
then added. After |
ca. |
1 min, |
the |
mixture |
||
was added to the resin and the reaction suspension gently agitated at room temperature for 24 h (note 7). The resin was then washed with DMF (10 min, 2.5 mL min 1) and Fmoc deprotected using 20% v/v piperidine in DMF (7 min, 2.5 mL min 1). The resin was then washed with DMF (10 min, 2.5 mL min 1), after which the excess DMF was removed. A solution of 4-methoxysulphonyl chloride (83 mg, 0.4 mmol) in DMF (1 mL) was added to the resin, followed by DIEA (26 mL, 0.15 mmol). The resultant suspension was gently agitated at room temperature for 24 h. The resin was then washed with DMF (10 min, 2.5 mL min 1); collected in a Buchner funnel; and successively washed with DMF (10 mL), DCM (20 mL), and hexane (5 mL); and dried in vacuo overnight. The amount of resin product recovered was 105 mg.
The derivatized resin product was suspended in DCM (6 mL) for 30 min, after which TFA (0.06 mL) was added; the resultant suspension was stirred for 10–15 min at ambient temperature (note 8). The suspension was filtered, the spent resin was washed with DCM (5 mL) and DCM:MeOH (1:1, 5 mL), and the combined filtrate was evaporated to dryness in vacuo to afford the title compound (28 mg, 90%). RP-HPLC analysis (G2) showed predominantly (>90%) N-(4-methoxy-benzenesulpho- nyl)-leucyl hydroxamic acid (Rt ¼ 10.4 min).
m / z (ES(þ)) calculated, 317.12 (MHþ ); observed, 317.3 (MHþ ).
H (250 MHz, CDCl3:[2H]6-DMSO) 0.70 (3H, d, J 6.3 Hz, Leu CH3), 0.83 (3H, d, J 6.4 Hz, Leu CH3), 1.37–1.60 (3H, m, Leu C H2 and C H), 3.74 (1H, m, Leu C H), 3.87 (3H, s,
Procedures 93
OCH3), 6.83 (1H, d, J 8.3 Hz, Leu NH), 6.96 (2H, d, J 8.8 Hz, Ar Hs), 7.46 (1H, s, NH), 7.79 (2H, d, J 8.8 Hz, Ar Hs).
H-D-Arg-Arg-Arg-Trp-D-Trp-Arg-Phe-NHOH
N-(Fmoc-Phe)-aminooxy-2-chlorotrityl polystyrene (88 mg, 0.60 mmol g 1, 0.0528 mmol), placed in a reaction column (note 9) was left in DMF (1 mL) for 18 h and then Fmoc-deprotected using 20% v/v piperidine in DMF (10 min, 2.5 mL min 1). The resin was then washed with DMF (10 min, 2.5 mL min 1), and the peptide sequence H-d-Arg(Pmc)-Arg(Pmc)-Arg(Pmc)-Trp(Boc)- d-Trp(Boc)-Arg(Pmc)- was assembled using the automated MilliGen PepSynthesizer 9050 (note 9).
Sequential acylation reactions were carried out at ambient temperature for 1.5 h using a DMF solution (1.3 mL) of the appropriate N-Fmoc–protected amino acids [Fmoc-Arg/d- Arg(Pmc)-OH, 265 mg; Fmoc-Trp/d-Trp(Boc)-OH, 211 mg; 0.4 mmol) and then carboxyl activated using TBTU (154 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol), and DIEA (140 mL, 0.8 mmol). Repetitive N -Fmoc deprotection was achieved using 20% v/v piperidine in DMF (6 min, 2.5 mL min 1).
The assembled N -Fmoc-deprotected peptidyl resin was collected in a Buchner funnel; washed with DMF (10 mL), DCM (20 mL), and MeOH (5 mL); and dried in vacuo overnight. The amount of resin product recovered 162 mg (0.0433 mmol).
The resin product was suspended in TFA (9 mL), into which
was immediately added water |
(0.45 mL), |
1,2-ethanedithiol |
(0.45 mL), and triisopropylsilane |
(0.1 mL). |
The mixture was |
left, with occasional agitation, at 30 C for 4 h. The suspension was then filtered, the spent resin washed with TFA (3 1 mL) and the combined filtrate was evaporated to dryness in vacuo. The residual material was then triturated with diethyl ether (10 mL) to give a white solid, which was filtered, washed with diethyl ether (3 10 mL), and dried in vacuo to afford the title compound
94 N-Fmoc-aminooxy-2-chlorotrityl Polystyrene Resin
(47 mg, 97%) as a white solid. Based upon RP-HPLC analysis (G2), the purity (note 10) is estimated to be 90%.
Rt ¼ 10.0 min; m / z (ES(þ)) calculated, 1177.65 (MHþ ); observed, 1177.9 (MHþ ).
NOTES
1.The use of an excess of Fmoc-Cl (1.5 Eq.) and / or stronger basic conditions typically promote significant formation of
the undesired bis-protected compound, N,O-bis-Fmoc-hydro- xylamine (m.p. 159.5–161 C; ES-MS, m / z 478.4 (MHþ ;
calculated, 478.17); silica-TLC (ethyl acetate:hexane, 1:1) Rf ¼ 0.64.
2.DCM is redistilled from calcium hydride and stored over molecular sieve. This reaction can be carried out in an ovendried round-bottomed flask (10 mL) or using the Quest 210 semiautomated synthesizer 5-mL reaction vessels.
3.Fmoc-NHOH is generally not very soluble in DCM; freshly redistilled THF (ca. 1 mL) may be added to aid dissolution.
4.This causes the resin to shrink and aids in the handling of resin material. As a result, the dried resin product must be preswollen in DCM:DMF (1:1), DCM:THF (1:1), or DCM for 24 h before use for solid-phase chemistry.
5.The resin substitution level is based on spectrophotometric determination of the Fmoc-derived chromophore liberated
upon treatment with 20% piperidine/DMF using 290 nm ¼ 5253 M 1 cm 1, which was used to calculate the percent
efficiency.
6.The Checkers found that the condensation reaction was variable and could range from 36 to 54% Fmoc-substitution
Notes 95
levels. More consistent results (57–78%) were obtained when the reaction was carried out using 5 Eq. Fmoc-NHOH in the presence of 5 Eq. DIEA. Moreover, this alternative approach was found to be reliable when the reaction was performed on a larger scale (1.25 mmol); the resin product gave a Fmoc substitution of 0.70 mmol g 1.
7.Owing to steric hindrance, the acylation reaction must be carried out using a large excess (4–10 Eq.) of the activated acid and for an extended period. In some cases, repeat acylation is recommended. Acylation has also been success-
fully carried out using Fmoc–amino acid fluorides (e.g., Fmoc-Phe-F4, 4 Eq. in the presence of DIEA, 1.1 Eq.; 18 h;
> 98% acylation efficiency). While acylation with unhindered activated carboxylic acids are achieved in > 98%, acylation with hindered carboxylic acids generally resulted in ca. 80% efficiencies.
8.Acidolytic treatment using DCM:hexafluoroisopropanol (1:1) for 2 h at ambient temperature afforded the hydroxamic acid in only 45% yield. However, it is worth noting that the tethered Fmoc-N(Pr)-O-2-chlorotrityl polystyrene, on treatment with similar acidolytic cocktail effected quantitative release of Fmoc-N(Pr)-OH.
9.An OMNI Fit (1.0 10.0 cm) reaction column was used. Alternatively, this can be carried out using either the Quest 210 semiautomated synthesizer or the Advanced ChemTech peptide synthesizer.
10.The purity of peptides obtained generally varies (50–90%) with the assembled peptide sequence. Owing to the protracted 90% TFA treatment, the major impurity usually observed is the acid-catalyzed decomposition product, peptidyl acid—the quantity of this undesired side product varies with peptide sequence and, particularly, the C-termi- nus amino acid residue.
96 N-Fmoc-aminooxy-2-chlorotrityl Polystyrene Resin
DISCUSSION
Naturally occurring pseudopeptidyl hydroxamic acids e.g., actinonin, foroxymithine, propioxatins, and matlystatin B5 and synthetic hydroxamic acids6 are potent and selective inhibitors of many important metalloproteases, including matrix metalloproteases, angiotensin-converting enzyme, endothelin-converting enzyme, and enkephalinases. Inhibition of these proteases, which house a zinc atom within the catalytic domain, is the result of the ability of the hydroxamic acid functionality to form a bidentate chelate with the zinc atom. The sheer numbers of these endogenous metalloproteases, which are involved in a diverse range of biologic processes suggest that these enzymes are valuable targets for inhibition within the context of therapeutic intervention.
Hence, the implication of combinatorial chemistry for high throughput generation of structurally diverse hydroxamic acids is self-evident. Several solid-phase approaches for their syntheses have been reported,1,7–11 the majority of which are based on the anchoring of N-hydroxyphthalimide onto an appropriate solid support. After hydrazine-mediated N-deprotection, N-acylation of the resin-bound hydroxylamine would yield the desired O- anchored hydroxamic acid, which is typically released by acidolysis.
In 1983, Prasad et al.12 first reported the condensation of chloromethyl polystyrene with N-hydroxyphthalimide to give the ester, hydrazinolysis of which yielded the desired resin-bound hydroxylamine. However, the sole purpose of this reagent was to react with, and hence extract ketones from, a complex steroidal mixture, and its use for the solid-phase synthesis of hydroxamic acids was not explored. Recently, the exploitation of the above solid-phase approach for the synthesis of hydroxamic acids was independently reported by three groups,7–9 all of which differ only in the method for the initial anchoring of N-hydroxyphtha- limide to an 4-alkoxybenzyl alcohol functionalized polystyrene or trityl chloride polystyrene. Subsequent N-deprotection was
Discussion 97
achieved by prolonged treatment (12–18 h) with hydrazine hydrate in DMF to afford the key intermediate O-anchored hydroxylamine.
In contrast, we reported a facile and efficient method for the preparation of the key intermediate, aminooxy-2-chlorotrityl polystyrene, via the readily synthesized N-(9-fluorenylmethoxy- carbonyl)-hydroxyamine.1 The compound Fmoc-NHOH was synthesized, in excellent yield as a white crystalline solid, by reacting hydroxylamine with stoichiometric amount of Fmoc-Cl under mild basic conditions for 3–4 h. Using the high loading 2- chlorotrityl chloride polystyrene,2 Fmoc-NHOH was selectively O-anchored, via a simple SN1 reaction, to afford the desired N-(9- fluorenylmethoxycarbonyl)aminooxy-2-chlorotrityl polystyrene. Typically, this condensation reaction was achieved in efficiency > 90%. Selective O-anchoring is achieved owing to the steric bulk of the trityl moiety. Conversely, it is worth noting that in our subsequent studies, condensation of Fmoc-NHOH with substituted benzhydryl chloride polystyrene gave a mixture of O- and N-anchored derivatives.
Moreover, during the course of our studies, N-[1-(4,4-di- methyl-2,6-dioxocyclohex-1-ylidene)ethyl]hydroxylamine, DdeNHOH was also successfully coupled with 2-chlorotrityl chloride polystyrene in excellent efficiency.1 The novel compound Dde-NHOH was prepared, in 51% yield, by reacting 2-acetyldi- medone with hydroxylamine in MeOH:THF at 5 C for 3 h, followed by recrystallization from ice-cold hexane; the major side-product, which increases in quantity over prolonged reaction time, was the predicted cyclized derivative 3,6,6-trimethyl-4-oxo- 4,5,6,7-tetrahydro-1,2-benzisoxazole.
N-(9-Fluorenylmethoxycarbonyl)aminooxy-2-chlorotrityl polystyrene was then N-deprotected within minutes by treatment with 20% v/v piperidine in DMF to afford the key intermediate aminooxy-2-chlorotrityl polystyrene. With this in hand, N- acylation was then carried out and, where appropriate, followed by a series of chemical transformations to yield resin-bound
98 N-Fmoc-aminooxy-2-chlorotrityl Polystyrene Resin
hydroxamic acid derivatives; examples of these transformations were illustrated above.
In this synthetic strategy, release of the assembled resinbound hydroxamic acid derivatives was efficiently achieved by exposure of the resin material to mild acidic reagents, including 1% v/v TFA in DCM for 10–15 min. Although we have had limited success, acidolytic release of the assembled molecule could also be effected by exposure to 50% v/v HFIP in DCM for 2 h. It is noteworthy that the use of mild acidolytic reagents in our solid-phase strategy is a significant advantage, because strong acidic reagents are known to cause decomposition of hydroxamic acids to the corresponding acids.
In conclusion, we anticipate that N-Fmoc-aminooxy-2- chlorotrityl polystyrene will prove an indispensable reagent for the solid-phase synthesis of hydroxamic acids by multiple and combinatorial approaches. Not only is its production both efficient and cost effective, but release of the assembled hydroxamic acid derivative is readily accomplished using mild acidolytic reagents.
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2.Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Peptide Protein Res. 1991, 37, 513.
3.Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
4.Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; DeSelms, R. H. J. Am. Chem. Soc. 1990, 112, 9651.
5.Umezawa, H.; Aoyagi, T.; Tanaka, T. et al. J. Antibiotics 1985, 38, 1629; Umezawa, H.; Aoyagi, T.; Ogawa, K. et al. J. Antibiotics 1985, 38, 1813; Inaoka, Y.; Takahashi, S.; Kinoshita, T. J. Antibiotics 1986, 39, 1378; and Tamaki, K.; Ogita, T.; Tanazawa, K.; Sugimura, Y. Tetrahedron Lett. 1993 34, 683.
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10.Ngu, K.; Patel, D. V. J. Org. Chem. 1997, 62, 7088.
11.Mellor, S. L.; Chan, W. C. Chem. Commun. 1997, 2005.
12.Prasad, V. V. K.; Warnes, P. A.; Lieberman, S. J. Steroid Biochem. 1983, 18, 257.